The present invention relates in general to a novel non-pathogenic microbe that produces a nontoxic, non-antigenic exopolysaccharide. The use of the microbe and exopolysaccharide in environmental engineering, agricultural, geologic, consumer and medical applications is described. Inhibition and control of mucoidy exopolysaccharide is also described.
The invention pertains to a novel non-pathogenic microbe that produces a non-toxic, non-antigenic exopolysaccharide. The exopolysaccharide can be used as a biofilm in environmental engineering and agricultural applications and as a filler or polymer in consumer and medical applications. Biofilm applications are described first, then particular medical applications are described.
The term xe2x80x9cbiofilmxe2x80x9d is used to describe an organic material that includes microorganisms embedded in a polymer matrix of their own making. The matrix consists largely of exopolysaccharides and is a tough, elastic, mucoidal material that adheres strongly to soil particles. Growth of a biofilm in a sandy soil is achieved by injecting a bacterial and nutrient solution into soil specimens. The resulting biofilm treatment is used to clog soil pores, thereby reducing the ability of the soil to transmit fluids.
Examples of biofilms are produced by certain strains of Klebsiella pneumoniae and Pseudomonas species. A problem with the use of K. pneumoniae is that Klebsiella is a genus that includes a number of human pathogens. Furthermore, the pathogenicity of K. pneumoniae itself is associated with its ability to create a mucoidal exopolysaccharide used in attachment and colonization that helps the pathogen evade both the non-specific and specific immune clearing defensive mechanisms.
Another example of a biofilm is described in U.S. Pat. No. 4,800,959, by Costerton, which discloses the use of a microbial process for selectively plugging a subterranean formation. In the process taught, a highly permeable stratum or zone in a subterranean reservoir is plugged using Klebsiella or Pseudomonas bacteria that were starved to reduce their size prior to being injected into the target zone. The bacteria regain full cell size, proliferate and commence production of biofilm-forming exopolysaccharides upon exposure to minimal nutrient containing media. The biofilm produced by these bacteria selectively seal off the high permeability zones of a formation and reduce aqueous flow through the zone.
In addition to the above described biofilm uses, there has been a need for perfusion solutions and blood substitutes. Currently available and approved compounds, however, have so far failed to meet the increasing demands on our blood provider system. A number of blood substitutes have been developed over the last few years to attempt to meet the increasing demand for blood, blood substitutes and plasma expanders. Unfortunately, many of the plasma expanders that are currently in use fail as the small molecules on which they depend to provide osmotic pressure readily traverse capillary beds as a consequence of the negative osmotic pressure found in post-arterial capillary beds. The loss of osmotic potential, makes the long-term use of current plasma expanders for maintaining proper ionic or fluid balance or plasma volume in a mammalian subject unsatisfactory.
Those blood substitutes that have an impermeable substance to maintain volume use human serum albumin or a mixture of plasma proteins as the oncotic agent. These substitute plasma proteins depend on the same blood and plasma supply as our current blood provider system, therefore failing to meet the increased demand for these products.
A number of patents have issued to Segall that are directed to blood and plasma substitutes. U.S. Pat. No. 4,923,442, and the reissue thereof, discloses a number of solutions used in blood substitution of living subjects all of which include at least some concentration of a cardioplegia agent, usually potassium ion. U.S. Pat. No. 4,923,442 discloses surgical methods, particularly in respect to instrument placement and the control of pulmonary wedge pressure generally applicable to perfusion of subjects. U.S. Pat. No. 5,130,230 discloses a blood substitute that may be used as a system of solutions in which a number of solutions, are used sequentially to completely replace the blood of living subjects. U.S. Pat. No. 5,130,230 discloses that the blood substitute comprises xe2x80x9can aqueous solution of electrolytes at physiological concentration, a macromolecular oncotic agent, a biological buffer having a buffering capacity in the range of physiological pH, simple nutritive sugar or sugars, and magnesium ion in a concentration sufficient to substitute for the flux of calcium across cell membranes.xe2x80x9d
In addition to the patented inventions described above, a number of commercially available products have been used for the treatment of hypovolemic patients. These include: HESPAN(trademark) (6% hetastarch in 0.9% sodium chloride injection, PENTASPAN(trademark) (10% pentastarch in 0.9% sodium chloride injection [both by DUPONT PHARMACEUTICALS(trademark), Wilmington Del.]), MACRODEX(trademark) (6% dextran 70 in 5% dextrose injection or 6% dextran 70 in 0.9% sodium chloride injection [PHARMACIA, INC.(trademark), Piscataway, N.J.]) and RHEOMACRODEX(trademark) (10% dextran 40 in 5% dextrose injection or 10% dextran 40 in 0.9% sodium chloride injection [PHARMACIA, INC.(trademark), Piscataway, N.J.]). All of these products, however, depend on compounds that are polymeric and that often dissociate or are broken down by natural physiologic enzymes with time. Alternatively, bacteria may take advantage of these newly supplied nutrient sources, causing severe septicemia in patients that are infected by pathogens at the time of injury. Thus, a need remains for a better oncotic agent.
The ability to produce mucoidal exopolysaccharides in medically important bacteria is critical to attachment to surfaces, resulting in increased resistance to drug treatments. Both chemical and physical treatments have been developed to control biofilm formation. Methods in Enzymology, Vol. 310, Biofilms, Ed. Ron J. Doyle, Academic Press, 1999. However, because biofilms are associated with pathogenicity, persistent and resistant bacterial infections and bio-corrosion of industrial structures, there is need for additional simple and efficient methods to control biofilms.
The newly discovered bacterium LAB-1, deposited at ATCC No. PTA-2500, possesses a number of potential commercial biofilm applications. These include, but are not limited to: (1) subsurface biofilm cutoff wall formation; (2) subsurface liners that include compacted, biofilm treated soil; (3) in-situ biofilm liners; (4) barriers made by treating geotextiles with biofilm materials; (5) improved ability of sand to retain moisture; (6) reclamation of poor soils and conversion into agriculture land; (7) significantly increased soil biomass in the form of polymers that function as a nutrient supply for plant growth and/or help retain nutrients and water; and (8) providing cohesion to otherwise cohesionless soils (such as sand dunes), thus making the soil more resistant to erosion by wind and/or water.
It has been found that the prior art methods and biofilms fail to provide biologically and environmentally safe and efficacious water, soil and waste retention characteristics. A significant problem with existing technology is the pathogenicity of the bacteria used to produce the biofilms. The present invention, therefore, is directed to a non-pathogenic bacterium that produces a biofilm made of exopolysaccharide that is essentially made of neutral sugars that migrate at the same rate as: mannose, fucose, fructose and galactose, acidic sugars that migrate at the same rate as fucose and amine sugars that migrate at the same rate as glucose and fucose.
More particularly, the bacterium is a LAB-1 strain. The biofilm producing bacterium may be further defined as being capable of growth between about pH 4 and 11 and between about 15xc2x0 and 45xc2x0 C. The LAB-1 strain is capable of growth in minimal growth media, or may be grown in an aqueous nutrient medium that includes yeast, peptone and mineral salt ingredients. LAB-1 is a gram-negative, rod-shaped bacterium of about 0.2xc3x970.8 xcexcm that secretes the exopolysaccharide described herein.
In one embodiment of the present invention, the LAB-1 strain is used in plugging a permeable subterranean stratum by providing LAB-1 bacteria in a nutrient-containing solution into the target stratum. The nutrient-containing solution is generally adapted to provide substantial and uniform growth conditions for the LAB-1. Sufficient biofilm is produced under these conditions to effectively plug the stratum. For example, the bacterium in situ can yield a saturated hydraulic conductivity equal to or less than 1.5xc3x9710xe2x88x92 cm/sec, equal to or less than 1.0xc3x9710xe2x88x92 cm/sec or even equal to or less than 1.5xc3x9710xe2x88x92 cm/sec.
Alternatively, the bacteria may be preincubated in culture in an aqueous suspension medium with agitation for an incubation period sufficient to initiate bacterial exopolysaccharide production before injection into the stratum. The method of plugging the subterranean stratum may also include draining nutrient deficient suspension medium from the reservoir, and recharging the reservoir with aqueous nutrient medium to maintain bacterial growth for an elapsed time period sufficient to establish a biofilm of prescribed saturated hydraulic conductivity. The draining and recharging steps with aqueous nutrient medium may be conducted at least once every 48 hours of elapsed time period. The step of pre-incubating the bacteria may be, e.g., for at least about 72 hours. These growth conditions permit for the establishment of a biofilm having a population between about 10-10 bacterial Colony Forming Units per square centimeter on a slide surface.
The biofilm may be used to plug open conduits, deposited in a subsurface biofilm cutoff wall, used to enhance the water retaining ability of subsurface liners or even for improving the water retention capabilities of compacted, semi-compacted or loosened biofilm treated soil. When used in a liner, the biofilm may be deposited in-situ. The biofilm may also be used along with and/or to enhance environmental barriers by treating geotextiles with the biofilm.
Another important aspect of this polymer is its lack of antigenicity and toxicity in an animal system. This suggests several consumer/medical applications, including: (1) use a food additive or food thickening or filler agent; (2) use as plasma expander; (3) use in polymer industry; (4) use as chromatography matrix support for purification of chemicals; (5) use in scientific research as suspension solution instead of ficoll and the like; (6) use in determining the gene content of the organism, especially those coding for the biosynthesis of the exopolysaccharide polymer; (7) use of the polymer materials in the cosmetic field; (8) use to augment insect or animal diets; (9) use as an additive in tissue culture media; (10) for use as a semi-solid to solid matrix, e.g, gel electrophoresis; (11) for use as an additive in toothpaste, ointments, creams and lotions; (12) for mixing with dyes, stains, paints and varnishes; (13) for inclusion in dialysis; (14) for use in composite materials, e.g., bricks, tile, mortars; (15) for use as part of a sealant; (16) viscosity modifier for oils, waxes and greases; (17) use as a filler, thickener or extender in pharmaceutical preparations; (18) use of the polymer in bioscaffolding applications, including wound-healing applications; and (19) use as a bacteriostatic (biostat) agent to inhibit or at least fail to support bacterial growth, and even possibly as a biocide.
In particular, a compound is needed for use as a plasma extender that serves to increase blood volume and that is impermeable at blood capillaries. The compound must not readily dissociate or be rapidly broken down by natural physiologic enzymes with time. Furthermore, the compound and its use as a plasma expander must not provide bacteria with an exogenous nutrient source, which may lead to accentuating already severe septicemia in patients that are infected by pathogens at the time of the injury that is causing hypovolemia.
More particularly, the present invention is an exopolysaccharide produced by the LAB-1 bacterial stain. The exopolysaccharide does not appear to easily support bacterial growth. This was determined by testing the ability of E. coli or S. indica to grow on the exopolysaccharide and no growth was observed. Further, the exopolysaccharide is not antigenic as tested by injection into mice. Thus, the product appears to satisfy some of the basic parameters required for a plasma expander.
The exopolysaccharide is secreted into the cell culture medium and collected for use in, e.g., a plasma expander. When used as a plasma expander alone, or in combination with other elements, the exopolysaccharide will be provided in an isotonic solution. In one embodiment, a blood-free plasma expander and blood substitute for use in a subject in need thereof includes a single solution with at least two water soluble oncotic agents, one of which is a water soluble polysacoharide oncotic agent and one of which is serum albumin, wherein the exopolysaccharide consisting essentially of mannose, fucose, fructose and galactose, acidic fucose and amine containing glucose and fucose.
The plasma expander and blood substitute may have a ratio of water soluble exopolysaccharide oncotic agent to serum albumin between 1:1 and 1:2, weight to weight. The combined percentage of water-soluble exopolysaccharide oncotic agent and serum albumin in a solution of the plasma expander and blood substitute may be in the range of between about 4%-6% weight to volume.
The plasma expander and blood substitute may also include a number of cations, alone or in combination. For example, the cations may be provided in the following concentrations: Na+at 110 to 120 mEq/1, Ca++at about 5 mEq/1, K+at 0 to 3 mEq/1, and Mg++at 0 to 0.9 mEq/1. These nations may be supplied as dissolved chloride salts. The plasma expander and blood substitute may also include at least one buffer, for example, a lactate and/or bicarbonate buffer. When buffered, the plasma expander will generally be a biological buffer having a buffering capacity in the pH range of about 6.8 to 7.8.
When used in hypovolemic patients, e.g., those that have lost a large volume of blood due to trauma, additional agents may be included in the plasma expander to aid in recovery. Such agents may include, Vitamin K in a concentration of about 1-4 mg/l, amylase, clotting factors, t-PA or even erythropoietin.
In non-medical uses, the exopolysaccharide of the present invention may be used as a chromatography matrix support for purification of chemicals. One such use will be as a suspension solution for use in centrifugation. The exopolysaccharide may even be used in solution as a suspension solution for use in size separation.
The present invention may also be used as a biologically stable, non-toxic material for use in coated plates for a number of biological and analytical uses. Examples of such uses include the coating of tissue culture plates for maintaining the growth, in vitro, of cells. Cells that may be grown on the surface of the exopolysaccharide include prokaryotic and eukaryotic cells. In an analytical setting, the exopolysaccharide disclosed herein may be used as a coating for instrumentation, such as biosensors, that require the maintenance of a biologically compatible environment.
Compositions containing propionic acid and ibuprofen when incorporated into liquid or solid growth media of the LAB-1 strain at a concentration range of 0.1-1.0% (w/v) differentially inhibits its growth, development, cell attachment and biofilm production. Growth of the newly discovered LAB-1 strain as well as its production of mucoidal exopolysaccharide and biofilm may be inhibited or controlled by prodionic acid, derivatives of propionic acid, compounds with related chemical structures or backbones such as 2-(4-isobutylphenyl)-propionic acid, otherwise known as ibuprofen, and solutions, mixtures, suspensions and other kinds of preparations comprising such compounds singly or in combination with other materials and compounds. It would be apparent to one of ordinary skill in the art to apply the above methods to inhibit the production of mucoid compounds and biofilm in any mucoid organism.
A more complete appreciation of the present invention and the scope thereof can be obtained from the accompanying drawings which are briefly summarized below, the following detailed description of the presently-preferred embodiments of the invention, and the appended claims.